DNA sensor-associated type I interferon signaling is increased in ulcerative colitis and induces JAK-dependent inflammatory cell death in colonic organoids.

DNA sensor pathways can initiate inflammasome, cell death, and type I interferon (IFN) signaling in immune-mediated inflammatory diseases (IMIDs), including type I interferonopathies. We investigated the involvement of these pathways in the pathogenesis of ulcerative colitis (UC) by analyzing the expression of DNA sensor, inflammasome, and type I IFN biomarker genes in colonic mucosal biopsy tissue from control (n = 31), inactive UC (n = 31), active UC (n = 33), and a UC single-cell RNA-Seq dataset. The effects of type I IFN (IFN-ß), IFN-γ, and TNF-α on gene expression, cytokine production, and cell death were investigated in human colonic organoids. In organoids treated with cytokines alone, or in combination with NLR family pyrin domain-containing 3 (NLRP3), caspase, or JAK inhibitors, cell death was measured, and supernatants were assayed for IL-1ß/IL-18/CXCL10. The expression of DNA sensor pathway genes-PYHIN family members [absent in melanoma 2 (AIM2), IFI16, myeloid cell nuclear differentiation antigen (MNDA), and pyrin and HIN domain family member 1 (PYHIN1)- as well as Z-DNA-binding protein 1 (ZBP1), cyclic GMP-AMP synthase (cGAS), and DDX41 was increased in active UC and expressed in a cell type-restricted pattern. Inflammasome genes (CASP1, IL1B, and IL18), type I IFN inducers [stimulator of interferon response cGAMP interactor 1 (STING), TBK1, and IRF3), IFNB1, and type I IFN biomarker genes (OAS2, IFIT2, and MX2) were also increased in active UC. Cotreatment of organoids with IFN-ß or IFN-γ in combination with TNFα increased expression of IFI16, ZBP1, CASP1, cGAS, and STING induced cell death and IL-1ß/IL-18 secretion. This inflammatory cell death was blocked by the JAK inhibitor tofacitinib but not by inflammasome or caspase inhibitors. Increased type I IFN activity may drive elevated expression of DNA sensor genes and JAK-dependent but inflammasome-independent inflammatory cell death of colonic epithelial cells in UC.NEW & NOTEWORTHY This study found that patients with active UC have significantly increased colonic gene expression of cytosolic DNA sensor, inflammasome, STING, and type I IFN signaling pathways. The type I IFN, IFN-ß, in combination with TNF-α induced JAK-dependent but NLRP3 and inflammasome-independent inflammatory cell death of colonic organoids. This novel inflammatory cell death phenotype is relevant to UC immunopathology and may partially explain the efficacy of the JAKinibs tofacitinib and upadacitinib in patients with UC.

Exercise and associated dietary extremes impact on gut microbial diversity.

OBJECTIVE: The commensal microbiota, host immunity and metabolism participate in a signalling network, with diet influencing each component of this triad. In addition to diet, many elements of a modern lifestyle influence the gut microbiota but the degree to which exercise affects this population is unclear. Therefore, we explored exercise and diet for their impact on the gut microbiota. DESIGN: Since extremes of exercise often accompany extremes of diet, we addressed the issue by studying professional athletes from an international rugby union squad. Two groups were included to control for physical size, age and gender. Compositional analysis of the microbiota was explored by 16S rRNA amplicon sequencing. Each participant completed a detailed food frequency questionnaire. RESULTS: As expected, athletes and controls differed significantly with respect to plasma creatine kinase (a marker of extreme exercise), and inflammatory and metabolic markers. More importantly, athletes had a higher diversity of gut micro-organisms, representing 22 distinct phyla, which in turn positively correlated with protein consumption and creatine kinase. CONCLUSIONS: The results provide evidence for a beneficial impact of exercise on gut microbiota diversity but also indicate that the relationship is complex and is related to accompanying dietary extremes.

Divergent metabolic outcomes arising from targeted manipulation of the gut microbiota in diet-induced obesity.

OBJECTIVE: The gut microbiota is an environmental regulator of fat storage and adiposity. Whether the microbiota represents a realistic therapeutic target for improving metabolic health is unclear. This study explored two antimicrobial strategies for their impact on metabolic abnormalities in murine diet-induced obesity: oral vancomycin and a bacteriocin-producing probiotic (Lactobacillus salivarius UCC118 Bac(+)). DESIGN: Male (7-week-old) C57BL/J6 mice (9-10/group) were fed a low-fat (lean) or a high-fat diet for 20 weeks with/without vancomycin by gavage at 2 mg/day, or with L. salivarius UCC118Bac(+) or the bacteriocin-negative derivative L. salivarius UCC118Bac(-) (each at a dose of 1×10(9) cfu/day by gavage). Compositional analysis of the microbiota was by 16S rDNA amplicon pyrosequencing. RESULTS: Analysis of the gut microbiota showed that vancomycin treatment led to significant reductions in the proportions of Firmicutes and Bacteroidetes and a dramatic increase in Proteobacteria, with no change in Actinobacteria. Vancomycin-treated high-fat-fed mice gained less weight over the intervention period despite similar caloric intake, and had lower fasting blood glucose, plasma TNFα and triglyceride levels compared with diet-induced obese controls. The bacteriocin-producing probiotic had no significant impact on the proportions of Firmicutes but resulted in a relative increase in Bacteroidetes and Proteobacteria and a decrease in Actinobacteria compared with the non-bacteriocin-producing control. No improvement in metabolic profiles was observed in probiotic-fed diet-induced obese mice. CONCLUSION: Both vancomycin and the bacteriocin-producing probiotic altered the gut microbiota in diet-induced obese mice, but in distinct ways. Only vancomycin treatment resulted in an improvement in the metabolic abnormalities associated with obesity thereby establishing that while the gut microbiota is a realistic therapeutic target, the specificity of the antimicrobial agent employed is critical.

Identification of an initiator-like element essential for the expression of the tissue inhibitor of metalloproteinases-4 (Timp-4) gene.

We have used real-time quantitative reverse transcriptase PCR (TaqMan) to quantify the expression of the four tissue inhibitor of metalloproteinases (Timp) genes in mouse tissues during development and in the adult. Among the four Timp genes, Timp-4 shows the most restricted pattern of expression, with highest RNA levels in brain, heart and testes. These data indicate that in the brain, Timp-4 transcripts are temporally regulated during development, becoming more abundant than those of the other Timps after birth. Cloning of the Timp-4 gene confirmed a five-exon organization resembling that of Timp-2 and Timp-3, and like all Timps, Timp-4 is located within an intron of a synapsin gene. Ribonuclease protection analysis and 5'-rapid amplification of cDNA ends PCR identified multiple transcription starts for Timp-4 from brain and heart mRNA. The promoter region of Timp-4 was functional in transient transfection analysis in mouse C3H10T1/2 fibroblasts, where it directed basal expression that was non-inducible by serum. The TATA-less promoter contains consensus motifs for Sp1 and an inverted CCAAT box upstream of an initiator-like element that is in close proximity to a transcription start site. Mutation of the CCAAT box caused a 2-fold increase in reporter expression. More significantly, mutation of the Sp1 motif or initiator-like element almost completely abolished reporter expression. This first functional characterization of the Timp-4 promoter shows it to be distinct from other members of the Timp family and provides insights into potential mechanisms controlling the tight spatio-temporal expression pattern of the gene.

Metalloproteinase expression in PMA-stimulated THP-1 cells. Effects of peroxisome proliferator-activated receptor-gamma (PPAR gamma) agonists and 9-cis-retinoic acid.

The PPAR gamma agonists, thiazolidinediones (TZDs), have anti-inflammatory properties as well as increasing insulin sensitivity. This has widened their therapeutic scope to treat inflammatory diseases such as atherosclerosis in addition to Type 2 Diabetes. TZDs are known to reduce monocyte/macrophage expression of Matrix metalloproteinase (MMP)-9, which is implicated in atherosclerotic plaque destabilization. This study aims to identify other metalloproteinase genes of the ADAM (A Disintegin And Metalloproteinase) and ADAMTS families that are regulated by PPAR gamma or RXR agonists, which are potentially important in type 2 diabetes and/or related atherosclerosis. The synthetic PPAR gamma agonist, GW7845, and the natural agonist 15d-PGJ2, suppressed PMA stimulated MMP-9 in human monocyte-like cells (THP-1) only in the presence of 9-cis-retinoic acid. Quantitative Real-Time PCR showed that this reduction was regulated at the mRNA level. Expression of ADAMs 8, 9, and 17 were increased, and ADAM15 was decreased by stimulation of THP-1 with PMA, although these ADAMs were not regulated by PPAR gamma or RXR agonists. PMA-induced ADAM28 expression was further enhanced by the addition of 9-cis-retinoic acid. ADAMTS4, implicated in rheumatoid arthritis, was expressed in THP-1 cells, and significantly increased after 24 h of PMA stimulation. ADAMTS4 expression was suppressed by both PPAR gamma and RXR agonists and was undetectable when the agonists were combined. Pretreatment of THP-1 cells with the PPAR gamma antagonist, GW9662, suggests that PPAR gamma plays subtly different roles in the regulation of MMP-9, ADAMTS4 and ADAM28 gene expression. These results indicate that PPAR gamma and RXR agonists have complex effects on monocyte metalloproteinase expression, which may have implications for therapeutic strategies.